Recent strategies to reprogram cell lineage have the potential to transform in vitro disease modeling, drug screening, and gene and cell therapies for regenerative medicine. There has been a recent expansion in cell reprogramming methods following the discovery of reprogramming of somatic cells to pluripotency by overexpression of master transcription factors. However, it still remains a pertinent challenge to generate cell types with functionally mature phenotypes at high efficiency. To address this bottleneck, we are developing new strategies to identify and relieve barriers to cell reprogramming. We propose to utilize next-generation epigenome editing tools based on the programmable CRISPR/Cas9 system to modulate the endogenous epigenome, discover barriers to reprogramming and phenotypic maturation, and facilitate reprogramming outcomes. In this project, we will use CRISPR/Cas9-based epigenetic modifiers to remodel endogenous chromatin at genes encoding master regulatory factors to direct differentiation of human pluripotent cells and reprogram human fibroblasts to induced neurons. We will use this strategy to induce reprogramming with transient delivery of the synthetic epigenetic modifiers, thus avoiding any genetic manipulation of the starting cell population. We will then exploit the high-throughput capacity of the CRISPR/Cas9 system for (1) the unbiased identification of transcription factor combinations that maximize production of induced neurons and (2) the identification of key regulatory elements that govern neuronal cell fate specification. The insights gained from these studies will have broad relevance to improving cell reprogramming strategies and enhancing our understanding of cell differentiation and plasticity.